Pharmaceutical composition and use thereof for relieving progression of chronic kidney disease or preventing thereof

ABSTRACT

The current invention is in the field of molecular biology/pharmacology and provides methods of using a pharmaceutical composition of 5-(2′,4′-difluorophenyl)-salicylanilide derivatives and their ring-fused analogs for preventing, inhibiting, reducing, or treating chronic kidney disease, conditions leading to or arising from it, and/or negative effects of each thereof.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition. In particular, the present invention relates to a pharmaceutical composition for relieving progression of chronic kidney disease or preventing thereof.

BACKGROUND OF THE INVENTION

Worldwide, chronic kidney disease (CKD), also known as chronic renal disease, is becoming a common disease in the general population. CKD is evaluated in terms of overall renal function (glomerular filtration rate, GFR) and the presence of kidney damage established by either kidney biopsy or other markers of kidney damage. CKD is a progressive loss in renal function over a period of months or years. The pathogenic mechanisms that lead to CKD converge on similar results in progressive renal inflammation and fibrosis.

In CKD, renal fibrosis progresses gradually and irreversibly, leading to an eventual loss of renal functions. Renal fibrosis, characterized by glomerulosclerosis and tubulointerstitial fibrosis, is the common manifestation of a wide variety of CKD. The pathogenesis of renal fibrosis is, in essence, a monotonous process that is characterized by an excessive accumulation and deposition of extracellular matrix (ECM) components. CKD patients are diagnosed with end-stage renal disease when the condition progresses to a point where only 15% or less of renal functions remains. Devastating disorder of CKD requires dialysis or kidney transplantation.

For example, chronic glomerulonephritis is a common cause of end-stage renal disease (ESRD), and most patients require renal replacement therapy to survive. IgA nephropathy (IgAN) and lupus nephritis (LN) is the most common primary and secondary glomerular diseases, respectively, and tend to affect young adults, with nearly 20% of patients progressing to ESRD after 10 years because of limited drug treatment options.

On the other hand, renal tubulointerstitial lesions (TILs) characterized by combined inflammation and fibrosis in the kidney are key pathological findings underlying the progression to end-stage renal disease

In view of these problems, it is an urgent matter to develop new, safe therapies to relieving progression of chronic kidney disease, and/or preventing chronic kidney disease. In other words, there is a need for the novel application of new drugs or of new indications for existing molecules and compounds

SUMMARY OF THE INVENTION

An object of the invention is to provide an agent/a medicament/a pharmaceutical composition for relieving progression of chronic kidney disease.

Another aspect of the present invention provides methods of preventing chronic kidney disease, and pharmaceutical compositions for use in treating chronic kidney disease in subjects such as those with IgA nephropathy, lupus nephritis, or renal tubulointerstitial lesions.

In summary, these and other objects have been achieved according to the present disclosure which demonstrates the pharmaceutical composition attenuatimg glomerular cell proliferation, attenuating sclerosis and fibrosis, attenuating neutrophil infiltration, attenuatings mononuclear leukocyte infiltration, attenuating crescent formation, attenuating fibrinoid necrosis, downregulating the expression of NLRP3 or/and IL-1β in a kidney of the CKDs' subject.

Detailed description of the invention is given in the following embodiments with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.

FIG. 1A-1O depict clinical assessments and renal pathology in IgAN mice. FIG. 1A-1C, 1G, 1H-1K demonstrate P-IgAN mouse model. FIG. 1D-1F, 1L, 1M-1O demonstrate S-IgAN mouse model. FIG. 1A and FIG. 1D represent Serum levels of BUN. FIG. 1B and 1E represent Serum levels of Cr. FIG. 1C and 1F represent Urine albumin/Cr. FIG. 1G and 1L represent H&E staining. FIG. 1H-1K and 1M-1O represent Scoring of H&E staining. Scale bars=50μm. Original magnification x400. Black arrows indicate mononuclear leukocyte infiltration. The arrowhead indicates neutrophil infiltration in glomeruli. Data are shown as the mean ±SD of 7 mice per group. P-WT+Saline, C57BL/6 mice injected with saline only; P-IgAN+Vehicle, passively induced IgAN mice treated with vehicle (PEG 400) only; P-IgAN+LCC18, passively induced IgAN mice treated with LCC18; S-WT, age-matched BALB/c mice; S-IgAN+Vehicle, spontaneous IgAN in gddY mice (S-IgAN) treated with vehicle only; S-IgAN+LCC18, spontaneous IgAN mice treated with LCC18; BUN, blood urea nitrogen; Cr, creatinine. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. #Not detectable. ns, no significant difference. Data of FIG. 1A-1B, 1D-1E, 1H-1K, 1M-1O were analyzed using ANOVA (with Dunnett's multiple comparisons test). Data of FIG. 1C, 1F were analyzed using t-tests (two-tailed).

FIG. 2A-2N depict NLRP3 inflammasome activation in IgAN mice and inhibition in IgAN mice via administrated LCC18. FIG. 2A-2C, FIG. 2G and FIG. 2H-2J demonstrate P-IgAN mouse model. FIG. 2D-2F, 2K and FIG. 2L-2N demonstrate S-IgAN mouse model. FIG. 2A and FIG. 2D represent Renal levels of ROS. FIG. 2B and 2E represent Serum levels of IL-1(3. FIG. 2C and 2F represent renal caspase-1 activity. FIG. 2G-2N represent renal levels of NLRP3, IL-1β and caspase-1. Data are shown as the mean ±SD of 7 mice per group. P-WT+Saline, age-matched C57BL/6 mice injected with saline only; P-IgAN+Vehicle, passively induced IgAN mice treated with vehicle (PEG 400) only; P-IgAN+LCC18, passively induced IgAN mice treated with LCC18; S-WT, age-matched BALB/c mice; S-IgAN+Vehicle, spontaneous IgAN in gddY mice (S-IgAN) treated with vehicle only; S-IgAN+LCC18, spontaneous IgAN mice treated with LCC18. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. ns, no significant difference. Data of FIG. 2A-2F, 2H-2J, 2L-2N were analyzed using ANOVA (with Dunnett's multiple comparisons test).

FIG. 3A-3J depict that LCC18 inhibits activation of DCs and systemic immune responses in IgAN model. (FIG. 3A) The fluorescence intensity of CD80⁺in gated CD11c⁺ cells (within gated CD11c⁺cells, grey-filled area created by staining with an isotype-matched control antibody) determined by flow cytometry. (FIG. 3B) TNF-α secretion measured by ELISA in BMDCs from C57BL/6 were treated for 0.5 h with or without LCC18 and then incubated for 24 h with or without IgA ICs. (FIG. 3C) T-cell proliferation measured by [³H]thymidine and secretion of (FIG. 3D) IFN-γ measured by ELISA as indicated at a 1:4 ratio of BMDCs: T cells for 3 days. BMDCs were incubated for 24 h with IgA ICs and co-cultured with OT-II CD' T cells pulsed with OVA peptide. P-IgAN mouse model (FIG. 3E-3G). S-IgAN mouse model (FIG. 3H-J). CD3 T-cell proliferation in splenocytes by [³H]thymidine uptake (FIG. 3E and 3H). Numbers of CD4±CD44^(hi)CD62L^(lo) (FIG. 3F and 3I) and CD8⁺CD44^(hi)CD62L^(lo-hi) (FIG. 3G and J) memory T cells among splenic cells assessed by flow cytometry. WT, wild type. #Not detectable. BMDCs, bone marrow-derived dendritic cells. Bars show the mean ±SD of 7 mice per group. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. ns, no significant difference. Data of FIG. 3B-3J were analyzed using ANOVA (with Dunnett's multiple comparisons test).

FIG. 4A-4B illustrate LPS-binding protein and IL-1β levels in SLE patients. (FIG. 4A) Serum levels of LPS-binding protein. (FIG. 4B) IL-1β secretion from PBMCs treated with LPS and ATP, followed by treatment with LCC18. Data are presented as means ±SEM. HC, healthy control; SLE, systemic lupus erythematosus; SS, Sjögren syndrome; PBMCs, peripheral blood mononuclear cells. **p<0.01, *** p<0.005, ****p<0.001. Data of FIG. 4A-4B were analyzed using ANOVA (with Dunnett's multiple comparisons test).

FIG. 5A-5N illustrate renal function, albuminuria, pathology, IgG anti-dsDNA, and cytokines in ASLN mice. Serum levels of (FIG. 5A) BUN and (FIG. 5B) Cr; (FIG. 5C) Urine albumin levels; (FIG. 5D) Renal pathology, H&E staining. Scale bars=50 μm. Original magnification 400×. (FIG. 5E-5I) Scoring for renal pathology; (FIG. 5J) Scoring for glomerulonephritis activity; (FIG. 5K) Serum levels of IgG anti-dsDNA autoantibody; Serum levels of (FIG. 5L) IL-1β, (FIG. 5M) IL-6 and (FIG. 5N) TNF-α. The arrows indicate mononuclear leukocyte infiltration. The arrowheads indicate neutrophil infiltration. Data are presented as means ±SEM for seven mice per group. ASLN, accelerated, severe lupus nephritis. BUN, blood urea nitrogen; Cr, creatinine. ***p<0.005, ****p<0.001, #Not detectable. Data of FIG. 5A-5C, 5E-5N were analyzed using ANOVA (with Dunnett's multiple comparisons test).

FIG. 6A-6M illustrate immunohistochemistry, flow cytometry in ASLN mice and OT-II antigen-specific T cell proliferation analysis. (FIG. 6A) F4/80⁺ macrophages infiltrating the kidney; (FIG. 6B) CD3+ T cells infiltrating the kidney; (FIG. 6C) CD11c⁺ dendritic cells infiltrating the kidney by IHC. Scale bars=50 p.m. Original magnification, 400x. The arrows indicate positive staining. (FIG. 6D-6I) Scoring for IHC; (FIG. 6J) CD4+CD44^(hi)CD62L^(lo) T memory cells; (FIG. 6K) CD8⁺CD44^(hi)CD62L ^(lo-hi)T memory cells; (FIG. 6L) CD4+CD25+Foxp3+ Treg cells. Flow cytometry using splenocytes. (FIG. 6M) T cell proliferation analysis using splenocytes by [H³]-thymidine incorporation assay. Data are presented as means ±SEM with seven mice per group. ASLN, accelerated, severe lupus nephritis. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. #Not detectable. ns, no significant difference. Data of FIG. 6D-6M were analyzed using ANOVA (with Dunnett's multiple comparisons test).

FIG. 7A-7J illustrate renal ROS production and NLRP3 inflammasome activation in ASLN mice. (FIG. 7A) ROS; (FIG. 7B) NF-kB activity; mRNA levels of NLRP3 (FIG. 7C), IL-1β (FIG. 7D), caspase-1(Fig. 7E); (FIG. 7F) Protein levels NLRP3, caspase-1 and IL-1β; (FIG. 7G-7I) Semiquantitative analysis of FIG. 7F. (FIG. 7J) Caspase-1 activity. Data are presented as means ±SEM with seven mice per group. ROS, reactive oxygen species; ASLN, accelerated, severe lupus nephritis. **p<0.01, ***p<0.005, ****p<0.001. Data of FIG. 7A-7E, 7G-7J were analyzed using ANOVA (with Dunnett's multiple comparisons test).

FIG. 8A-8D show that renal histopathological evaluation using hematoxylin and eosin (H&E) (FIG. 8A-8B) and Masson's trichrome staining (FIG. 8C-8D). Scoring of TILs (FIG. 8B) and renal tubulointerstitial fibrotic area (FIG. 8D). *p<0.05, ***p<0.005, ****p<0.001. ns, no significant difference. Data of FIG. 8B, 8D were analyzed using ANOVA (with Dunnett's multiple comparisons test).

FIG. 9 shows that F4/80+ macrophages infiltrating the kidney, CD3⁺ T cells infiltrating the kidney, Col-III and Samd2/3 in the kidney by IHC.

FIG. 10A-10B shows that ELISA analysis of urine protein levels of IL-1β (FIG. 10A) and MCP-1 (FIG. 10B) collected from the dilated pelvis. *p<0.05, ***p<0.005, ****p<0.001. ns, no significant difference. Data of FIG. 10A-10B were analyzed using ANOVA (with Dunnett's multiple comparisons test).

FIG. 10C shows Western blot analysis for IL-1β, caspase-1 and IL-36α of renal tissues of Saline control mice, UUO+Vehicle mice and UUO+LCC18 mice.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the invention are shown and described herein, such embodiments are provided by way of example only and are not intended to otherwise limit the scope of the invention. Various alternatives to the described embodiments of the invention may be employed in practicing the invention.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The present invention provides a method for treating, preventing, or alleviating a symptom of CKD by administering to the subject a pharmaceutical composition, or a pharmaceutically acceptable salt, prodrug, metabolite, solvate, and polymorph thereof, optionally in a therapeutically effective amount.

Optionally, in an exemplary embodiment of the present invention, the pharmaceutical composition of the present invention includes, but are not limited to, 5-(2′,4′-difluorophenyl)-salicylanilide derivatives and their ring-fused analogs.

Furthermore, the 5-(2′,4′-difluorophenyl)-salicylanilide derivatives include, but are not limited to, an 2-hydroxy-N-[3-(trifluoromethyl)phenyl]benzamide, N-(4-chloro-2-fluorophenyl)-2-hydroxybenzamide, N-(3-chloro-4-fluorophenyl)-2-hydroxybenzamide, and 6-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenyl)-2H-benzo[e] [ 1,3] oxazine-2,4(3 H)-dione.

In one embodiment, 6-(2,4-difluorophenyl)-3 -(3-(trifluoromethyl)phenyl)-2H-benzo[e] [1,3] oxazine-2,4(3 H)-dione is defined as LCC18.

A subject (patient) may be a human being or a non-human animal, such as cat, dog, rabbit, cattle, horse, sheep, goat, monkey, mouse, rat, gerbil, guinea pig, pig, but is preferably a human. Usually the individual has suffered or is in risk of developing CKD that results in some degree of kidney function loss and/or has a condition that will result in CKD. Preferably, the subject has suffered or is in risk of developing a CKD (such as a subject having diseases and conditions that can damage the kidneys). Diseases and conditions that can damage the kidneys and lead to CKD include, but are not limited to, autoimmune disorders (such as systemic lupus erythematosus).

In addition, the pharmaceutical composition of the present invention can relieve progression of or prevent CKD of the subject. The CKDs include, but are not limited to, glomerulonephritis, renal fibrosis, and renal inflammation.

For example, in some embodiments, the glomerulonephritis in the present invention include, but not limited to, primary glomerulonephritis and secondary glomerulonephritis.

On the other side, the primary glomerulonephritis in the present invention includes, but not limited to, IgA nephropathy. Furhermore, the secondary glomerulonephritis in the present invention includes, but not limited to, lupus nephritis. Moreover, the glomerulonephritis in the present invention includes, but not limited to, renal tubulointerstitial lesions.

The pharmaceutical composition of the present invention may be administered prior to, concurrently, or after the onset of physical or histological symptoms of CKD, such as the time of renal tissue injury, or the establishment of tubulointerstitial fibrosis.

The “therapeutic effective amount” used herein refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of the disorder, or prevent advancement of the disorder, or cause regression of the disorder without undesirable side effects, such as toxicity, irritation or allergic response. Although individual needs may vary, the determination of optimal ranges for effective amounts of formulations is within the skill of the art. Human doses can readily be extrapolated from animal studies (Katocs et al., Chapter 27 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990).

The term “therapeutically effective amount”, as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician. In a preferred aspect, the disease or condition to be treated is a chronic kidney disease, including chronic kidney disease in Stage 1, 2, 3, 4, and 5.

For example, with respect to the treatment of CKD, a therapeutically effective amount means an amount effective to treat CKD, which is an amount effective to reverse, halt, or delay the progress of CKD, or to confer protection of kidney from subsequent damage.

In one embodiment, a therapeutically effective amount will refer to the amount of a therapeutic agent that decreases the amount of deposition of extracellular matrix in the obstructed kidney by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. The detection of an alteration can be carried out in vitro e.g., using a biological sample or in vivo. A biological sample may be any tissue or fluid from a subject that is suitable for detecting the deposition of extracellular matrix. In one embodiment, the biological sample could be evaluated by a non-invasively detecting kit comprising RussiaSea-001 (Crenomytilus grayanus lectin/ CGL) or RussiaSea-002 (Mytilus trossulus lectin/ MTL).

In a further embodiment, a therapeutically effective amount will refer to the amount of a therapeutic agent that improves a patient's kidney function by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Kidney function may be assessed by any method known in the art, such as by measuring blood Creatinine levels, Creatinine clearance, or urine Creatinine levels.

Generally, the dosage required to provide an effective amount of a formulation, which can be adjusted by one skilled in the art, will vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s) (Nies et al., Chapter 3 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).

A “pharmaceutical composition” is a formulation containing the compounds of the present invention in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The dosage will depend on the route of administration via depending on the age and condition of the subject. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every day, every 2 days, every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

For example, in some embodiments, the pharmaceutical composition of the present disclosure is administered once per month, twice per month, three times per month, every other week, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily, twice a day, or three times a day.

Whilst the dosage of the pharmaceutical composition used will vary according to the activity and the condition being treated, it may be stated by way of guidance that a dosage selected in the range from 5 to 30 mg/kg per body weight per dose, particularly in the range from 10 to 20 mg/kg of body weight per dose.

EXAMPLES

The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

EXAMPLE 1

LCC18 improves renal function, albuminuria and renal pathology in IgAN animal models

Two complementary mouse models of IgAN — a passive IgAN (P-IgAN) and a spontaneous IgAN in gddY mice (S-IgAN) were used to examine therapeutic effects of LCC18 on renal conditions of IgAN. In other word, two complementary IgAN mouse models included: (1) a passively induced IgAN (P-IgAN) model, characterized by mesangial cell proliferation and scattered neutrophil infiltration in glomeruli. Eight-week-old female C57BL/6 mice were injected with IgA anti-PC intraperitoneally and PnC intravenously for 14 or 28 consecutive days. Treatment with LCC18 (daily dose of 10 mg/kg body weight) dissolved in polyethylene glycol 400 (PEG 400) (Sigma-Aldrich, St. Louis, MO, USA) via an intraperitoneal (IP) route (P-IgAN+LCC18), and another group of IgAN mice was administered vehicle only (PEG 400) as the disease control group (P-IgAN+Vehicle). Mice were killed at days 14 and 28. Age-matched C57BL/6 mice injected with saline (P-WT+Saline) were used as normal controls; (2) a spontaneous IgAN in gddY (S-IgAN) mice, featuring severe glomerular and tubulointerstitial lesions, characterized by mesangial proliferation, mesangial matrix expansion, and tubulointerstitial inflammation. LCC18 (S-IgAN+LCC18) or vehicle (S-IgAN+Vehicle) was administered to mice daily beginning at 8 weeks of age and continuing throughout the present invention.

The gddY mice were sacrificed at 32 weeks. Age-matched BALB/c mice served as normal controls (S-WT). All animal experiments were conducted with approval of the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taipei, Taiwan, and were conducted in accordance with NIH Guidelines for the Care and Use of Laboratory Animals.

For the P-IgAN model, the mice received daily at day 7 after disease induction, either LCC18 (10 mg/kg body weight) (P-IgAN+LCC18) or vehicle (P-IgAN+Vehicle) via IP throughout the study. For the S-IgAN model, LCC18 (S-IgAN+LCC18) or vehicle (S-IgAN+Vehicle) was administered to the mice daily throughout the study via IP, beginning at 8 weeks of age. Significantly lower serum levels of BUN and Cr were recorded (FIG. 1A, FIG. 1B) in P-IgAN+LCC18 mice than in P-IgAN mice treated with vehicle (P-IgAN+Vehicle mice), which showed increased levels of these parameters for renal function compared to those of C57BL/6 mice, wild type for P-IgAN, injected with saline (P-WT+Saline mice). In addition, S-IgAN+LCC18 mice also benefited from treatment with the LCC18 (FIG. 1D, FIG. 1E). In parallel, significantly decreased urine levels of albumin, demonstrated by the urine albumin/Cr ratio, were observed in P-IgAN+LCC18 mice compared to P-IgAN+Vehicle mice. This beneficial effect began on day 21 after disease induction and increased until day 28 when mice were sacrificed (FIG. 1C). This therapeutic effect was also observed in S-IgAN+LCC18 mice beginning on week 24 and with vehicle treatment beginning on week 8, continuing to week 32 during LCC18 treatment (FIG. 1F).

By light microscopy, although glomerular cell proliferation, neutrophil infiltration and sclerosis in glomeruli and mononuclear leukocyte infiltration into the interstitial tissue were observed in P-IgAN+Vehicle mice, the severity of renal lesions was markedly reduced in P-IgAN+LCC18 mice (FIG. 1G, FIG. 1H-1K). In

S-IgAN+LCC18 mice, similar renal lesion improvement was noted, except that there was no detectable glomerular neutrophil infiltration in either S-IgAN+Vehicle or the S-IgAN+LCC18 mice (FIG. 1L, FIG. 1M-1O).

EXAMPLE 2

LCC18 inhibits NLRP3 inflammasome in IgAN mice

Significantly reduced ROS levels in renal tissues were recorded in

P-IgAN+LCC18 and S-IgAN+LCC18 mice compared to P-IgAN+Vehicle and S-IgAN+Vehicle mice (FIG. 2A and 2D). Moreover, increased serum levels of IL-1β in P-IgAN+Vehicle mice and in S-IgAN+Vehicle mice was markedly reduced by LCC18 treatment (FIG. 2B and 2E). Furthermore, although renal caspase-1 activity was significantly increased in P-IgAN+Vehicle and S-IgAN+Vehicle mice compared with P-WT+saline and S-WT mice, caspase-1 activity was reduced in P-IgAN+LCC18 and S-IgAN+LCC18 mice (FIG. 2C and 2F). The present invention then examined whether treatment of P-IgAN and S-IgAN mice with LCC18 could inhibit production of NLRP3, IL-1β and caspase-1 in renal tissues. Renal levels of these proteins were significantly reduced in P-IgAN+LCC18 and S-IgAN+LCC18 mice, compared with P-IgAN+Vehicle and S-IgAN+Vehicle mice (FIG. 2G—J). Collectively, these examples demonstrated that LCC18 is involved in negatively regulating NLRP3 inflammasome.

EXAMPLE 3

LCC18 inhibits dendritic cells (DCs) and T cell functions in IgAN mice

Significantly decreased fluorescence intensity levels of CD11c⁺CD80⁺ (an activation marker for DCs) cells (FIG. 3A) and reduced secretion of TNF-α (a maturation marker for DCs) (FIG. 3B) were seen in LCC18-treated IgA immune complexes (IgA ICs)-stimulated, bone marrow-derived DCs (BMDCs), compared with normal controls (mock). Using an OVA-specific T cell proliferation assay, IgA ICs-stimulated BMDCs induced markedly increased CD4⁺ T cell proliferation (FIG. 3C) and IFN-γ secretion from CD4⁺ T cells (FIG. 3D), but these effects were inhibited by LCC18 treatment.

Moreover, compared with S-IgAN+Vehicle and P-IgAN+Vehicle mice, S-IgAN+LCC18 and p-IgA+LCC18 mice showed significantly decreased T cell proliferation, as demonstrated by [³H] thymidine uptake (FIG. 3E and 3H). Although P-IgAN+Vehicle mice exhibited a higher total number of CD4⁺CD44^(hi)CD62L^(lo) and CD8⁺CD44^(hi)CD62L^(lo-hi) memory T cells in the spleen than P-WT+Saline and S-WT mice (FIG. 3F, 3G), this increase was inhibited in P-IgAN+LCC18 mice. In parallel, increases in splenic cells numbers of CD4⁺CD44^(hi)CD62L^(lo) and CD8⁺CD44^(hi)CD62L^(lo-hi) memory T cells in S-IgAN+Vehicle mice were also inhibited in S-IgAN+LCC18 mice (FIG. 31, 3J). Collectively, these examples demonstrated that LCC18 is involved in negatively regulating DCs and T cell functions.

EXAMPLE 4

Increased serum LBP levels in SLE patients and reduced IL-1β secretion in PBMCs by LCC18 treatment

Serum samples were collected from 65 patients with SLE, 24 with Sjogren syndrome and 30 healthy controls, while whole blood samples were collected from 14 SLE patients for the preparation of peripheral blood mononuclear cells (PBMCs) in Ficoll-Hypaque (Amersham Pharmacia Biotech, NJ, USA). All participants gave written informed consent and the research plan was approved by the Ethics Committee of Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (approval number: 2-106-05-001).

Levels of LPS binding protein (LBP) (MyBiosouce, Vancouver, Canada) and IL-1β (R&D Systems, MN, USA) were measured using commercial ELISA kits.

Significantly, in SLE patient's serum LBP levels were increased, compared with those of Sjogren's syndrome and normal subjects (FIG. 4A). Furthermore, treatment with LCC18, a small molecule that shows potent immunomodulatory effects, resulted in significantly reduced levels of IL-1β secretion by LPS-primed PBMCs collected from SLE patients, compared with those of saline controls (FIG. 4B). These results suggest that episodes of bacterial infection and may contribute to deterioration of the patients tested and LCC18 may have therapeutic effects in systemic lupus erythematosus (SLE) and lupus nephritis (LN).

EXAMPLE 5

LCC18 reatment improves renal function and reduces albuminuria and renal pathology in accelerated, severe lupus nephritis (ASLN) model

To establish ASLN model mice, eight-week-old female NZB/WF1 mice (prior to autoantibody production) were given LPS (Sigma, MO, USA) (0.8 mg/kg body weight) twice a week by intraperitoneal injection. Seven days after the first injection of LPS, seven mice each received either LCC18 (10 mg/kg body weight) or vehicle (polyethylene glycol 400) (Sigma) daily via intraperitoneal injections. Another group of age-matched female NZB/WF1 mice were injected with saline and served as normal controls. All mice were sacrificed 5 weeks after induction of the disease. All animal experiments were performed in compliance with the NIH Guidelines for the Care and Use of Laboratory Animals with approval of the Institutional Animal Care and Use Committee of The National Defense Medical Center, Taipei, Taiwan.

Next, an ASLN model in NZB/W F1 mice, which mimics acute onset of severe LN in human, was validated potential therapeutic effects of LCC18 in the present invention. LN was induced in these mice by intermittent LPS injections to simulate SLE patients following bacterial or viral infections believed to act as environmental triggers. LCC18 was administered daily after the onset of the renal condition in ASLN mice (ASLN+LCC18 mice), while ASLN mice that received vehicle only (ASLN+Vehicle mice) were used as disease control mice.

Urine samples were collected for albuminuria assessment using the ratio of urine albumin/urine creatinine (Cr). Serum blood urea nitrogen (BUN) and Cr levels were determined.

In one embodiment, elevated serum levels of BUN and Cr were seen in ASLN+Vehicle mice, but these effects were significantly inhibited in ASLN+LCC18 mice (FIG. 5A and 5B). In parallel, ASLN+LCC18 mice exhibited greatly reduced albuminuria, compared with ASLN+Vehicle mice, which manifested overt albuminuria in comparison with saline control mice (FIG. 5C).

On the other hand, renal tissues were fixed in formalin buffer and paraffin-embedded and stained with hematoxylin and eosin (H&E). Scoring of renal pathology was evaluated, and glomerulonephritis activity was scored. In another embodiment, while ASLN+Vehicle mice developed serious pathological changes, including intrinsic cell proliferation, crescent formation, fibrinoid necrosis, neutrophil infiltration in the glomeruli, and periglomerular interstitial inflammation, these effects were significantly inhibited in ASLN+LCC18 mice (FIG. 5D-5I). ASLN+LCC18 mice had significantly lower glomerulonephritis activity scores than ASLN+Vehicle mice (FIG. 5J).

EXAMPLE 6

LCC18 treatment reduces levels of anti-dsDNA and cytokines in sera for ASLN model

Levels of anti-dsDNA antibody (Alpha Diagnostic International, TX, USA) and IL-1β, IL-6 and TNF-α (R&D Systems, MN, USA) were measured using commercial ELISA kits.

Greatly reduced serum anti-dsDNA levels were observed in ASLN+LCC18 mice, compared with increased serum anti-dsDNA levels in ASLN+Vehicle mice in relation to saline control mice (FIG. 5K). In parallel, ASLN+LCC18 mice showed significantly reduced serum levels of IL-1β, IL-6, and TNF-α compared with ASLN+Vehicle mice (FIG. 5L-5N).

EXAMPLE 7

LCC18 treatment inhibits renal immune cell infiltration, and regulates systemic T cell functions for ASLN model

In addition, Methyl Carnoy solution-fixed and paraffin-embedded renal tissues were stained with antibodies against F4/80+ (macrophages; Serotec, NC, USA) or CD3⁺ (pan T cell; Dako, Glostrup, Denmark), and frozen sections were stained with CD11c⁺ antibody (BioLegend, CA, USA). For scoring the number of F4/80⁺-, CD3⁺- or CD11c⁺-positive cells, quantitative image analysis software (Pax-it; Paxcam, IL, USA) was applied.

In one embodiment, treatment with LCC18 resulted in greatly decreased levels of mononuclear leukocyte infiltration by F4/80⁺ macrophages, CD3⁺ T cells, and CD11c⁺ DCs in renal tissues of ASLN+LCC18 mice, compared with those of ASLN+Vehicle mice, as demonstrated immunohistochemically (FIG. 6A-6I). Moreover, LCC18 treatment inhibited CD4⁺ and CD8⁺ T cells (FIG. 6J-6K), but enhanced Treg cell differentiation (FIG. 6L) and T cell proliferation (FIG. 6M) in splenocytes from ASLN+LCC18 mice. These results suggest that differential regulation of T cell activation and Treg differentiation may be involved in the therapeutic effect of this small molecule in this ASLN mouse model.

EXAMPLE 8

LCC18 treatment reduces renal ROS and inhibits NLRP3 inflammasome activation for ASLN model

Reactive oxygen species (ROS) levels in renal tissues were measured by a lucigenin-enhanced chemiluminescence assay. Nuclear NF-KB p65 activity (Active Motif, Tokyo, Japan) and cytoplasmic caspase-1 activity (R&D systems) were measured, according to the manufacturer's instructions. Protein lysates from tissues and cultured cells were run on SDS-PAGE gels. Anti-NLRP3 (AdiopGen, CA, USA), caspase-1, β-actin (Santa Cruz, Tex., USA), IL-1β (R&D systems, MN, USA) antibodies were used for Western Blot analysis.

RNA was extracted by Trizol (Invitrogen) according to the manufacturer's instructions. Real-time PCR reactions were done using 10 μl of cDNA, 12.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems), and 1.25 pl of the specific probe/primer mixed in a total volume of 25 pl. The thermal cycler conditions were as follows: 2 min at 50° C., 10 min at 95° C., 40 cycles of denaturation (15s at 95° C.), and combined annealing/extension (1 min at 60° C.). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used as the internal standard. Sequences of all primers used are listed below (F: Forward primer; R: Reverse primer):

TABLE 1 The primers for quantitative real-time PCR. Primer/ probe name primer sequences  NLRP3 forward 5′-CTGTG TGTGG GACTG AAGCA C-3′ primer SEQ ID NO: 1 NLRP3 reverse 5′-GCAGC CCTGC TGTTT CAGCA-3′ primer SEQ ID NO: 2 IL-1β forward 5′-CCAGGATGAGGACATGAGCACC-3′ primer SEQ ID NO: 3 IL-1β reverse 5′-TTCTCTGCAGACTCAAACTCCAC-3′ primer SEQ ID NO: 4 Caspase-1 5′-ACTGTACAACCGGAGTAATACGG-3′ forward primer SEQ ID NO: 5 Caspase-1 5′-CACGGAAGGCCATGCCAGTGA-3′ reverse primer SEQ ID NO: 6 GAPDH forward 5′-TCCGCCCCTTCTGCCGATG-3′ primer SEQ ID NO: 7 GAPDH reverse 5′-CACGGAAGGCCATGCCAGTGA-3′ primer SEQ ID NO: 8 

ASLN+LCC18 mice exhibited significantly reduced renal levels of ROS, compared with ASLN+Vehicle mice (FIG. 7A). Additionally, significantly decreased renal NF-κB p65 activity was detected in ASLN+LCC18 mice compared with ASLN+Vehicle mice (FIG. 7B). Treatment with LCC18 significantly reduced mRNA levels of NLRP3, IL-1β, and caspase-1 in renal tissues of ASLN+LCC18 mice compared with ASLN+Vehicle mice (FIG. 7C-7E). In parallel, decreased protein levels of renal NLRP3, caspase-1 and IL-1β were observed in ASLN+LCC18 mice, compared with those of ASLN+Vehicle mice (FIG. 7F-7I). Also, ASLN+LCC18 mice showed significantly inhibited renal caspase-1 activity compared with ASLN+Vehicle mice (FIG. 7J).

EXAMPLE 9

LCC18 improves renal pathology in UUO mice mimicking renal tubulointerstitial lesions (TILs) via Histopathology

To establish Unilateral ureteral obstruction (UUO) model for mimicking renal tubulointerstitial lesions (TILs), the mice (8-12 weeks of age) were anesthetized followed by a lateral incision on the back of the mouse. After the left ureter was exposed, it was tied with a silk suture at two points and permanently ligated. Sham-operated mice, which underwent an identical procedure but without ureteric ligation, were used as sham control. After 7 or 14 days, the mice were euthanized, and then, renal tissues, pelvis urine, and renal draining lymph nodes were collected.

LCC18 (10 mg/ kg body weight) was administered intraperitoneally 1 day before (preventive group) or 3 days after (therapeutic group) the induction of UUO, or 1 day after the ligation of renal vessels in this model. Diseased mice that received only polyethylene glycol 400 (PEG 400) (Sigma-Aldrich, St. Louis, Mo., USA) daily via intraperitoneal injections were used as the vehicle control group. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taiwan, in compliance with the NIH Guide for the Care and Use of Laboratory Animals.

Renal tissues were fixed in 10% buffered formalin, embedded in paraffin, and 3-μm thick sections were cut for haematoxylin and eosin (H&E) staining. Fifty glomeruli were examined in at least two renal tissue sections per slide by light microscopy at ×400 magnification. The percentage of glomeruli showing proliferation, peri-glomerular inflammation, neutrophil infiltration, or glomerular sclerosis was determined.

The quantitative analysis of the data obtained from hematoxylin and eosin (H&E) and Masson's trichrome staining of renal histopathological evaluation was used by quantitative imaging software (Pax-it; Paxcam, Villa Park, Ill.). As shown in FIG. 8A-8D, while Vehicle+UUO mice exhibited renal inflammatory and fibrotic changes on both days 7 and 14, these findings were greatly improved in UUO+LCC18 mice at these time points.

EXAMPLE 10

LCC18 inhibits renal immune cell infiltration and improve renal fibrosis in UUO mice

Further, in another embodiment, formalin-fixed and paraffin-embedded renal sections were incubated with primary antibodies against mouse CD3 (BioLegend) and F4/80 (Serotec), followed by biotinylated secondary antibodies and avidin-biotin-peroxidase complex (Dako) for IHC staining.

Please refer FIG. 9, IHC shows infiltration of F4/80⁺ monocytes/macrophages and CD3+ T cells mainly in renal interstitial tissue of UUO+Vehicle mice on day 7 and the degree of these findings continued to enhance until dayl4, compared with sham control mice sham control (Sham-operated mice, which underwent an identical procedure but without ureteric ligation, were used as sham control). However, UUO+LCC18 mice showed significantly decreased infiltration of these inflammatory cells in the kidney on day 7 and 14, compared with UUO+Vehicle mice. In parallel, increased renal expression of Col-III, p-Smad2/3 and IL-36α was identified in UUO+Vehicle mice compared with sham control mice on days 7 and 14, but this effect was inhibited in UUO+LCC18 mice.

EXAMPLE 11

LCC18 inhibited NLRP3 inflammasome activation in UUO mice

In one embodiment, ELISAs for urine from the dilated pelvis of Sham control mice, UUO+Vehicle mice and UUO+LCC18 mice were performed using detection kits (all from R&D system, Minneapolis, Minn., USA). The levels of IL-1β and MCP-lwere detected by ELISA according to the manufacturer's instructions.

In another embodiment, renal tissues of Saline control mice, UUO+Vehicle mice and UUO+LCC18 mice were subjected to protein extraction using a kit (Millipore). Each of the target proteins of renal tissues was detected by SDS-PAGE and immunoblotting using antibodies against-IL-1β (R&D), caspase-1 (Adipogen), or IL-36α. (3-Actin (Santa Cruz) was used as an internal control.

In summary, the pharmaceutical composition attenuates glomerular cell proliferation about 15˜35%, attenuates sclerosis and fibrosis, attenuates neutrophil infiltration about 15˜50%, attenuates mononuclear leukocyte infiltration about 30˜60%, attenuates crescent formation about 40˜90%, attenuates fibrinoid necrosis about 40˜90% , downregulate the expression of NLRP3 or/and IL-1β in a kidney of the CKDs' subject.

Further, the pharmaceutical composition can regulate renal function and albuminuria, downregulate serum levels of blood urea nitrogen (serum levels of BUN) about 50˜60%, serum levels of creatinine (serum levels of Cr) about 30˜40% and serum levels of IgG anti-dsDNA about 60˜70% of the CKDs' subject.

The foregoing detailed description and examples have been provided for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described; many variations will be apparent to one skilled in the art and are intended to be included within the invention defined by the claims. 

What is claimed is:
 1. A pharmaceutical composition for relieving progression of or preventing chronic kidney disease in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition comprising a 5-(2′,4′-difluorophenyl)-salicylanilide derivatives and their ring-fused analogs.
 2. The pharmaceutical composition according to claim 1, wherein the 5-(2′,4′-difluorophenyl)-salicylanilide derivatives comprise an 2-hydroxy-N-I3-(trifluoroinethyl)phenylibenzamide, N-(4-chloro-2-f1uorophertyl)-2-hydroxybenzamide, N-(3-chloro-4-fluorophenyl)-2-hydroxybenzamide, and 6(2,4-difluorophenyl)-3-(3-(trifiuoromethyl)phenyl)-2H-benzo[e][1,3]oxazine -2,4(3H )-dione.
 3. The pharmaceutical composition according to claim 2, wherein the chronic kidney disease comprises glomerulonephritis, renal fibrosis, and renal inflammation.
 4. The pharmaceutical composition according to claim 3, wherein the glomerulonephritis is selected from primary glomerulonephritis, and secondary glomerulonephritis.
 5. The pharmaceutical composition according to claim 4, wherein the primary glomerulonephritis is selected from IgA nephropathy.
 6. The pharmaceutical composition according to claim 4, wherein the secondary glomerulonephritis is selected from lupus nephritis.
 7. The pharmaceutical composition according to claim 3, wherein the renal fibrosis is selected from renal tubulointerstitial lesions. 8 The pharmaceutical composition according to claim
 3. wherein the pharmaceutical composition attenuates glomerular cell proliferation about 15˜35%, attenuates sclerosis and fibrosis, attenuates neutrophil infiltration about 15˜50%, attenuates mononuclear leukocyte infiltration about 30˜60%, attenuates crescent formation about 40˜90%, attenuates fibrinoid necrosis about 40-90%, downregulate the expression of NLRP3 or/and IL-1β in a kidney of the subject.
 9. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition can regulate renal function and albuminuria, downregulate serum levels of blood urea nitrogen (serum levels of BUN) about 50˜60%, serum levels of creatinine serum levels of Cr) about 30˜40% and serum levels of IgG anti-dsDNA about 60˜70% of the subject.
 10. The pharmaceutical composition according to claim 3, wherein the therapeutically effective amount of the pharmaceutical composition is from about 5 mg/kg to about 30 mg/kg body weight per dose, wherein the subject is selected from mammals, wherein the mammals are selected from cat, dog, rabbit, cattle, horse, sheep, goat, monkey, mouse, rat, gerbil, guinea pig, pig and the human. 